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Results for “Intracellular quantification”

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PubMed1988

Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.

Apolipoprotein A-I (apoA-I), the major protein component of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapoA-I) with a hexapeptide extension (Arg-His-Phe-Trp-Gln-Gln) at its amino terminus. To investigate the regulation of processes that regulate plasma apoA-I levels, a sensitive and simple assay for proapoA-I is required. We describe a specific enzyme-linked immunosorbent assay (ELISA) for quantification of proapoA-I using monospecific rabbit antibodies raised against the peptide: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The monospecificity of antibodies to propeptide has been checked and no cross-reaction with mature apoA-I has been found although three first mature apoA-I amino acids (Asp-Glu-Pro) were included in the immunizing peptide. The assay is a non-competitive sandwich ELISA in which polystyrene microtiter plates were used with antibodies to propeptide adsorbed on the wells. After incubation with plasma samples, the bound proapoA-I was revealed by labeled rabbit polyclonal antibodies directed against mature apoA-I. The working range was 10 to 100 ng/ml, recovery of proapoA-I added to plasma was 94.6 to 106.5%, and the intra- and interassay coefficients of variation were 3.8% and 7.9%, respectively. A delipidation step using diisopropylether-n-butanol was necessary to expose antigen sites of proapoA-I in native lipoproteins. Mean level of proapoA-I in normal subjects was 87 +/- 15 micrograms/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%.

Apolipoprotein A-I↗

PubMed1994

Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy.

Numerous experiments on CHO cells have shown that endosomes are composed of separate vesicular and tubulovesicular compartments, such as the sorting endosome, the recycling compartment, and the late endosome. However, Hopkins et al. (Nature 346, 335-339, 1990) have reported that endosomes in HEp2 human carcinoma cells form an extensive tubular reticulum. To resolve their observations with previous results from CHO and other cells, we examined the sorting and intracellular transport of endocytosed macromolecules in HEp2 cells, using low density lipoprotein (LDL) and transferrin (Tf) to probe the lysosomally directed and recycling pathways, respectively. Fluorescent LDL and Tf were observed with laser scanning confocal microscopy to visualize simultaneously both probes' sorting and subsequent post-sorting behavior in HEp2 cells. Quantifying the 3-dimensional cellular distributions of fluorescent LDL and Tf, after a variety of pulsechase schemes, gave the ligands' trafficking rates. Initially, both ligands appear in the same punctate sorting endosomes, and fingers of Tf start extending from these sorting endosomes. Tf rapidly leaves dual-labeled sorting endosomes (t1/2 approximately 2.5 minutes) and enters a post-sorting recycling compartment from which it is recycled out of the cell (t1/2 approximately 7 minutes). We present both morphological and kinetic data supporting the existence of these two separate compartments along the recycling pathway in HEp2 cells. LDL remains in punctate sorting endosomes that eventually lose the ability to receive newly endocytosed LDL, and mature into late endosomes. The trafficking and sorting of Tf and LDL in HEp2 cells follow the same general scheme as in CHO cells, indicating that the tubular endosomes previously seen may be the tubular parts of the sorting endosomes and recycling compartments in these cells. We propose that the endosomes in the recycling pathway of HEp2 cells, as in CHO cells, are composed of short-lived sorting endosomes, accessible to both Tf and LDL, and long-lived post-sorting recycling compartments, which contain Tf and recycling receptors but not LDL.

Biological Transport↗

PubMed2005

Evaluation of magnesium fluxes in rat erythrocytes using a stable isotope of magnesium.

The mechanisms that maintain intracellular Mg concentration at physiologic levels are not fully understood. In this work, we described for the first time, a new method using 25Mg stable isotopes, that allows simultaneous determination of Mg2+ efflux and Mg2+ influx in non-loaded cells at physiological levels of extracellular Mg. Erythrocytes from rats were suspended as a 10% suspension in NaCl medium or choline medium. The erythrocyte suspension was incubated at 37C, and aliquots of the cell suspension were centrifuged at the beginning of the incubation and after 60 and 120 min. The quantification of 24Mg, 25Mg and 26Mg in supernatants and in erythrocytes were determined by ICP/MS. Simultaneous Mg2+ efflux and Mg2+ influx were calculated from the intra-extracellular distribution of the three isotopes. By this new approach we characterized Mg2+ influx and Mg2+ efflux at 0.4 mM extracellular Mg in both NaCl and choline Cl medium. Mg2+ efflux and Mg2+ influx were largely inhibited by amiloride in NaCl medium and by cinchonine in choline Cl medium. Apparent velocity and LineWeaver-Burk kinetics showed that Mg2+ influx is different from Mg2+ efflux suggesting the involvement of two distinct transport mechanisms. Moreover, modifying extracellular Mg concentrations, to mimic hypo- or hyper-magnesaemia, we showed that Mg2+ efflux and Mg2+ influx increased with extracellular Mg up to 0.8 mM, the physiologic concentration of total extracellular Mg. Our data demonstrate that Mg2+ fluxes are directly related to the levels of extracellular Mg and that in physiological conditions, Na-dependent and Na-independent Mg2+ efflux counterbalance Mg influx to maintain constant intracellular Mg level.

Animals↗

PubMed2001

Cooking oil fume-induced cytokine expression and oxidative stress in human lung epithelial cells.

Epidemiological studies have shown an association between exposure to indoor air pollution from Chinese-style cooking and risk of lung cancer among Chinese females. Several toxic substances have been identified in cooking oil fumes (COF) collected from heated rapeseed oil. In this study, we examined the biological effects of COF on CL3 human lung epithelial cells. Exposure to 200 microg/ml COF significantly reduced cell growth within 4 days. In addition, we examined the effect of COF on TGFbeta1, TGFbeta2, IL-6, IL-8, and IFN-gamma gene expressions with the RT-PCR method. We found that TGFbeta1 mRNA levels increased after exposure to 200 microg/ml COF for 24 h. Similarly, exposure to 10 microM benzo[a]pyrene or 100 nM 12-O-tetradecanoylphorbol-13-acetate increased TGFbeta1 mRNA levels at 24 h. The mRNA levels of TGFbeta2, IL-6, IL-8, and IFN-gamma did not increase after treatment with COF, benzo[a]pyrene, or 12-O-tetradecanoylphorbol-13-acetate. COF-induced TGFbeta1 production was confirmed by quantification of TGFbeta1 in conditioned medium with enzyme-linked immunosorbent assay. Exposure to 200 microg/ml COF significantly increased TGFbeta1 secretion in a time-dependent and dose-dependent manner. It has been demonstrated that reactive oxygen intermediates induce TGFbeta1 gene expression. When CL3 cells were exposed to 200 microg/ml COF for 15 min, there was an increase in intracellular peroxide formation with the dichlorofluorescein method. Furthermore, treatment with 200 microg/ml COF for 12 h also significantly induced lipid peroxidation in CL3 cells. Our results show that exposure to COF inhibits cell growth, increases TGFbeta1 secretion, and induces oxidative stress in CL3 lung epithelial cells. This suggests that TGFbeta1 and oxidative stress play a role in the biological effects of COF on lung epithelial cells.

Air Pollution, Indoor↗

PubMed2002

Pregnenolone metabolites in rat testis: endogenous concentrations, and intracellular distribution in whole testes during incubation in vitro.

The intracellular movements of pregnenolone in rat testes were investigated. Whole testes were incubated in the presence or absence of pregnenolone (2.5mM) in the medium for 120 min (in some studies 30, 60, and 90 min). The testes were homogenised, subcellular fractions prepared and analysed in quadruplicate for steroid content by gas chromatography-mass spectrometry with selected ion monitoring. Quantification of pregnenolone and 11 of its metabolites, obtained from non-incubated whole testes, provided values for endogenous amounts. Pregnenolone was the only steroid of quantitative importance found initially in the mitochondrial fraction but was subsequently found in the microsomal fraction, where metabolism occurred. Identification and quantification of metabolites indicated that both classical pathways for testosterone production were operating, with the 4-en-3-oxosteroid pathway predominating. By 120 min, virtually all pregnenolone metabolites, including pregnenolone itself, were found in the cytosol, consistent with an overall movement from mitochondria to endoplasmic reticulum to cytosol.

Animals↗

PubMed2003

Tissue transglutaminase mRNA expression in apoptotic cell death.

INTRODUCTION: Tissue transglutaminase (t.TG) is an enzyme that catalyzes the cross-linking of intracellular proteins, thus assembling a protein scaffold that prevents leakage of intracellular components. t.TG is activated during the apoptotic cell death cascade and plays a key role in the formation of apoptotic bodies. The aim of this study was to determine to what amount t.TG-mRNA becomes expressed during apoptosis and whether the t.TG-mRNA expression level could be used as trace marker of recent apoptosis and in individual cases for quantification of apoptosis. METHODS: Expression of t.TG-mRNA was determined using TaqMan based, real-time RT-PCR, a semi-quantitative RT-PCR technique. The t.TG-mRNA expression was measured in cultured cells (MCF-7, human endothelial cells) and in peripheral blood mononuclear cells (PBMCs) before and after induction of apoptosis in vitro. RESULTS: The TaqMan RT-PCR of t.TG proved to be reliable, reproducible (CV's inter and intraassay precisions of 0.8-2.8%, measured at two levels), and specific for apoptotic cell death. t.TG-mRNA expression increases in response to apoptosis induction and is not expressed during the process of necrotic cell death. The expression during apoptotic cell death changes in the dose dependent manner in cultured cells as well as in the PBMCs, treated in vitro. The increase t.TG-mRNA expression level was up to 20 times, depending on the intensity of the apoptosis induction treatment and incubation time afterwards. PBMCs of patients with myelodysplasia showed spontaneous expression of t.TG-mRNA in agreement with their increased apoptotic cell death in vivo. CONCLUSION: t.TG-mRNA expression increases significantly in response to apoptosis inducing treatment. The observed changes are dose and time dependent. This leads to the conclusion that t.TG expression can be used as a trace marker for detection and quantification of apoptosis.

Apoptosis↗

PubMed1992

Immunologic alterations in a murine model of hemorrhagic shock.

OBJECTIVE: To study multiple immune parameters in mice subjected to severe hemorrhage without fluid resuscitation. STUDY DESIGN: Controlled animal study. Anesthetized, female mice were hemorrhaged by tail bleeding. Immune parameters (spleen T-cell proliferation and activation, intracellular calcium flux, cytokine production, peritoneal neutrophil respiratory burst, and survival after intra-abdominal septic challenge) were measured at 24, 48, and 72 hrs after hemorrhage. MEASUREMENTS AND MAIN RESULTS: T-cell proliferation was decreased in animals after two 20% blood volume hemorrhages, 30 mins apart; single 30%, 40%, and 50% blood volume hemorrhages did not depress proliferation. "Helper/inducer" T cells from twice-hemorrhaged mice showed decreased expression of activation antigens (interleukin-2 receptor, Ia) after mitogen stimulation. In contrast, "suppressor/cytotoxic" T cells displayed increased activation, shown by augmented expression of interleukin-2 receptor and Ia antigens. Leukocyte production of prostaglandin E2, a mediator frequently implicated in immune down-regulation, was unaffected by hemorrhage. Secretion of tumor necrosis factor-alpha (TNF-alpha) in culture was increased when cells were harvested 48 hrs after injury. Intracellular calcium flux in stimulated lymphocytes was decreased 24 hrs after hemorrhage, suggesting deranged intracellular signal transduction. Respiratory burst activity of peritoneal neutrophils was unchanged following hemorrhage. When animals were subjected to septic challenge, the survival rate was markedly decreased after two hemorrhages (when sepsis was induced 24 hrs after hemorrhage). By 72 hrs posthemorrhage, most of the immunologic alterations, including resistance to septic challenge, had resolved. CONCLUSIONS: This uninstrumented hemorrhagic shock model allows quantification of multiple immune derangements. Immune suppression was identified after two smaller (20% blood volume) hemorrhages, but not after a single, larger hemorrhage. Immune derangements are maximal at 24 hrs posthemorrhage, and resolve in the subsequent 48 hrs.

Analysis of Variance↗

PubMed2001

Y2, the smallest of the Sendai virus C proteins, is fully capable of both counteracting the antiviral action of interferons and inhibiting viral RNA synthesis.

An open reading frame (ORF) overlapping the amino-terminal portion of the Sendai virus (SeV) P ORF in the +1 frame produces a nested set of carboxy-coterminal proteins, C', C, Y1, and Y2, which are referred to collectively as the C proteins. The C proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (IFNs), inhibit viral RNA synthesis, and are involved in virus assembly. In this study, we established HeLa cell lines stably expressing the C, Y1, and Y2 proteins individually and examined the capacities of these cells to circumvent the antiviral action of alpha/beta IFN (IFN-alpha/beta) and IFN-gamma and to inhibit viral transcription. The assay protocols included monitoring of IFN-alpha/beta-mediated signaling by interferon-stimulated response element-driven reporter gene expression and of the antiviral state induced by IFN-alpha/beta and IFN-gamma and measurement of reporter gene expression from an SeV minigenome, as well as quantification of SeV primary transcripts. When necessary, the activities measured were carefully normalized to the expression levels of the respective C proteins in cells. The data obtained clearly indicate that the smallest protein, Y2, was as active as the C and Y1 proteins in both counteracting the antiviral action of IFNs and inhibiting viral transcription. The data further show that intracellular transexpression of either C, Y1, or Y2 rendered HeLa cells moderately or only poorly permissive for not only wild-type SeV but also 4C(-) SeV, which expressed none of the four C proteins. On the basis of these findings, the roles of SeV C proteins in the natural life cycle are discussed.

Antiviral Agents↗

PubMed2004

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

Acid Phosphatase↗

PubMed2001

Possible existence of common internalization mechanisms among arginine-rich peptides.

Basic peptides such as human immunodeficiency virus type 1 (HIV-1) Tat-(48-60) and Drosophila Antennapedia-(43-58) have been reported to have a membrane permeability and a carrier function for intracellular protein delivery. We have shown that not only Tat-(48-60) but many arginine-rich peptides, including HIV-1 Rev-(34-50) and octaarginine (Arg(8)), efficiently translocated through the cell membranes and worked as protein carriers (Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) J. Biol. Chem. 276, 5836-5840). Quantification and time course analyses of the cellular uptake of the above peptides by mouse macrophage RAW264.7, human cervical carcinoma HeLa, and simian kidney COS-7 cells revealed that Rev-(34-50) and Arg(8) had a comparable translocation efficiency to Tat-(48-60). Internalization of Tat-(48-60) and Rev-(34-50) was saturable and inhibited by the excess addition of the other peptide. Typical endocytosis and metabolic inhibitors had little effect on the internalization. The uptake of these peptides was significantly inhibited in the presence of heparan sulfate or chondroitin sulfates A, B, and C. Treatment of the cells with the anti-heparan sulfate antibody or heparinase III also lowered the translocation of these peptides. These results strongly suggest that the arginine-rich basic peptides share a certain part of the internalization pathway.

Amino Acid Sequence↗

PubMed1996

Effect of astroglial cell swelling on pH of acidic intracellular compartments.

A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH4Cl (2 mM) and chloroquine (10 microM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (-80 mOsm) and treatment of the cells with glutamate (l mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio (485/440 nm) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 +/- 0.07, 0.85 +/- 0.14 and 0.25 +/- 0.07, respectively. Measurement of cytosolic pH using 2',7',-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 +/- 0.05 pH units) and 1 mM glutamate (by 0.26 +/- 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.

Acridine Orange↗

PubMed2007

A whole-body model to distinguish excess fluid from the hydration of major body tissues.

BACKGROUND: Excess fluid (ExF) accumulates in the body in many conditions. Currently, there is no consensus regarding methods that adequately distinguish ExF from fat-free mass. OBJECTIVE: The aim was to develop a model to determine fixed hydration constants of primary body tissues enabling ExF to be calculated from whole-body measurements of weight, intracellular water (ICWWB), and extracellular water (ECWWB). DESIGN: Total body water (TBW) and ECWWB were determined in 104 healthy subjects by using deuterium and NaBr dilution techniques, respectively. Body fat was estimated by using a reference 4-component model, dual-energy X-ray absorptiometry, and air-displacement plethysmography. The model considered 3 compartments: normally hydrated lean tissue (NH_LT), normally hydrated adipose tissue (NH_AT), and ExF. Hydration fractions (HF) of NH_LT and NH_AT were obtained assuming zero ExF within the diverse healthy population studied. RESULTS: The HF of NH_LT mass was 0.703 +/- 0.009 with an ECW component of 0.266 +/- 0.007. The HF of NH_AT mass was 0.197 +/- 0.042 with an ECW component of 0.127 +/- 0.015. The ratio of ECW to ICW in NH_LT was 0.63 compared with 1.88 in NH_AT. ExF can be estimated with a precision of 0.5 kg. CONCLUSIONS: To calculate ExF over a wide range of body compositions, it is important that the model takes into account the different ratios of ECW to ICW in NH_LT and NH_AT. This eliminates the need for adult age and sex inputs into the model presented. Quantification of ExF will be beneficial in the guidance of treatment strategies to control ExF in the clinical setting.

Adipose Tissue↗

PubMed2006

Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons.

Intracellular Ca2+ regulates a variety of neuronal functions, including neurotransmitter release, protein phosphorylation, gene expression and synaptic plasticity. In a variety of cell types, including neurons, Ca2+ is involved in actin reorganization, resulting in either actin polymerization or depolymerization. Very little, however, is known about the relationship between Ca2+ and the actin cytoskeleton organization in retinal neurons. We studied the effect of high-K+-induced depolarization on F-actin organization in salamander retina and found that Ca2+ influx through voltage-gated L-type channels causes F-actin disruption, as assessed by 53 +/- 5% (n = 23, P < 0.001) reduction in the intensity of staining with Alexa-Fluor488-phalloidin, a compound that permits visualization and quantification of polymerized actin. Calcium-induced F-actin depolymerization was attenuated in the presence of protein kinase C antagonists, chelerythrine or bis-indolylmaleimide hydrochloride (GF 109203X). In addition, phorbol 12-myristate 13-acetate (PMA), but not 4alpha-PMA, mimicked the effect of Ca2+ influx on F-actin. Activation of ionotropic AMPA and NMDA glutamate receptors also caused a reduction in F-actin. No effect on F-actin was exerted by caffeine or thapsigargin, agents that stimulate Ca2+ release from internal stores. In whole-cell recording from a slice preparation, light-evoked 'off' but not 'on' EPSCs in 'on-off' ganglion cells were reduced by 60 +/- 8% (n = 8, P < 0.01) by cytochalasin D. These data suggest that elevation of intracellular Ca2+ during excitatory synaptic activity initiates a cascade for activity-dependent actin remodelling, which in turn may serve as a feedback mechanism to attenuate excitotoxic Ca2+ accumulation induced by synaptic depolarization.

Actins↗

PubMed1998

Estimations of intra- and extracellular volume and pH by 31P magnetic resonance spectroscopy: effect of therapy on RIF-1 tumours.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

Animals↗

PubMed2002

Four cell types with distinctive membrane properties and morphologies in lamina I of the spinal dorsal horn of the adult rat.

Lamina I of the spinal dorsal horn plays an important role in the processing and relay of nociceptive information. Signal processing depends, in part, on neuronal membrane properties. Intrinsic membrane properties of lamina I neurons were therefore investigated using whole cell patch clamp recordings in a slice preparation of adult rat spinal cord. Based on responses to somatic current injection, four cell types were identified: tonic, which fire comparatively slowly but continuously throughout stimulation; phasic, which fire a high frequency burst of variable duration; delayed onset, which fire irregularly and with a marked delay to the first spike; and single spike, which typically fire only one action potential even when strongly depolarised. Classification by spiking pattern was further refined by identification of characteristic stimulus-response curves and quantification of several response parameters. Objectivity of the classification was confirmed by cluster analysis. Responses to stimulus trains and synaptic input as well as the kinetics of spontaneous synaptic events revealed differences in the signal processing characteristics of the cell types: tonic and delayed onset cells appeared to act predominantly as integrators whereas phasic and single spike cells acted as coincidence detectors. Intracellular labelling revealed a significant correlation between morphological and physiological cell types: tonic cells were typically fusiform, phasic cells were pyramidal, and delayed onset and single spike cells were multipolar. Thus, there are multiple physiological cells types in lamina I with specific morphological correlates and distinctive signal processing characteristics that confer significant differences in the transduction of input into spike trains.

Animals↗

PubMed1987

Quantitative analysis of a nuclear antigen in interphase and mitotic cells.

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.

Antigen-Antibody Complex↗

PubMed1997

Changes in nitric oxide metabolite levels in stimulated substantia nigra neurons.

Simultaneous quantification of the two major nitric oxide (NO) metabolites, nitrite and nitrate, was performed on mouse mesencephalic neurons in vitro, in basal conditions or after excitatory stimuli. Measurements were made using both extracellular medium and cytosolic extracts. Basal metabolite levels were stable up to 5 min. Depolarization by high levels of potassium promptly increased extracellular nitrite (270% of basal, peak at 10s) leaving intracellular levels unchanged. Glutamate receptor agonists caused a rapid increase in intracellular, but not extracellular, nitrite levels (240% of basal, peak at 30s). Intracellular nitrate levels raised slowly after addition of potassium (215% of basal at 1 min) or N-methyl-D-aspartate (230% of basal at 5 min), but not after quisqualate. The different dynamic variations of the two NO metabolites point to the specificity of the activated metabolism according to the type of excitatory stimulus.

Animals↗

PubMed2001

Modulation of lens cell adhesion molecules by particle beams.

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

Cell Adhesion↗